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41.
The three-dimensional structure of the human immunoglobulin fragment Fab New (IgG1, lambda) has been refined to a crystallographic R-factor of 16.9% to 2 A resolution. Rms deviations of the final model from ideal geometry are 0.014 A for bond distances and 3.03 degrees for bond angles. Refinement was based on a new X-ray data set including 28,301 reflections with F > 2.5 sigma(F) from 6.0 to 2.0 A resolution. The starting model for the refinement procedure reported here is from the Brookhaven Protein Data Bank entry 3FAB (rev. 1981). Differences between the initial and final models include modified polypeptide-chain folding in the third complementarity-determining region (CDR3) and the third framework region (FR3) of VH and in some exposed loops of CL and CH1. Amino acid sequence changes were determined at a number of positions by inspection of difference electron density maps. The incorporation of amino acid sequence changes results in an improved VH framework model for the "humanization" of monoclonal antibodies. 相似文献
42.
The packing of peptide helices in crystals of the leucine-rich decapeptide Boc-Aib-Leu-Aib-Aib-Leu-Leu-Leu-Aib-Leu-Aib-OMe provides an example of ladder-like leucylleucyl interactions between neighboring molecules. The peptide molecule forms a helix with five 5----1 hydrogen bonds and two 4----1 hydrogen bonds near the C terminus. Three head-to-tail NH ... O = C hydrogen bonds between helices form continuous columns of helices in the crystal. The helicial columns associate in an antiparallel fashion, except for the association of Leu ... Leu side chains, which occurs along the diagonal of the cell where the peptide helices are parallel. The peptide, with formula C56H102N10O13, crystallizes in space group P2(1)2(1)2(1) with Z = 4 and cell parameters a = 16.774(3) A, b = 20.032(3) A and c = 20.117(3) A; overall agreement factor R = 10.7% for 2014 data with magnitude of F(obs) greater than 3 sigma (F); resolution 1.0 A. 相似文献
43.
Summary The ectomycorrhizal fungus,Paxillus involutus, produces sclerotia in culture. These can be induced to form on agar medium by exposing mycelium grown at 25°C to various temperatures between6°C and 15°C. Sclerotia formed at 10°C and above were large and covered with drops of exudate, while those formed at 6°C or 8°C were very small and did not produce an exudate. Mature sclerotia were bounded by a compact rind and contained abundant storage reserves. Histochemistry of the larger sclerotia showed large quantities of protein stored as protein bodies in the cytoplasm, lipid present as small droplets, glycogen granules stored in the cytoplasm and polyphosphate present as small granules in the cytoplasm and in the protein bodies. Energy dispersive X-ray microanalysis confirmed the presence of phosphate in the granules and was used to map its distribution throughout the sclerotium. The smaller sclerotia induced at 8°C and below on the same medium had the same basic structure and composition, but lacked the complex phenolic cell network found in large sclerotia, and had abundant extracellular polysaccharides. The rind was not well developed and these small sclerotia are interpreted to have been arrested at an early stage of development. 相似文献
44.
Ion accumulation in the cell walls of rice plants growing under saline conditions: evidence for the Oertli hypothesis 总被引:10,自引:3,他引:7
Abstract. When plants of rice ( Oryza saliva L.) are subjected to mildly saline (50mol m−3 NaCl) conditions, the leaves show symptoms of water deficit, even though ion accumulation has been more than sufficient to adjust to the decrease in external water potential. After a few days of exposure to salt, there is a negative correlation, in a population of leaves, between the leaf water concentration (g water per g dry weight) and their sodium concentration (mmol Na per g dry weight). Ion concentrations in the cell walls and the cytoplasm of cells of plants grown in low salinity were measured by X-ray microanalysis. The NaCl concentration in solution in the apoplast was calculated to be around 600mol m−3 in leaves of plants whose roots were exposed to only 50 mol m−3 NaCl. This constitutes strong evidence that an important factor in salt damage in rice is dehydration due to the extracellular accumulation of salt as suggested in the Oertli hypothesis. The implication, that changes in tissue ion concentration and solute potentials equivalent to the external medium is not evidence of plant osmotic adjustment to salinity, is discussed. 相似文献
45.
Jean-Marc Gilli Paolo Mariani Franco Rustichelli Bin Yang Hervé Delacroix 《Molecular Engineering》1992,2(2):177-188
We recently showed that a side-chain industrial co-oligosiloxane presents a quenchable enlarged blue phase behaviour at the cholesteric-isotropic phase transition. In this paper, we present the results of a structural study based on X-ray diffraction, differential scanning calorimetry and optical measurements. In particular, the smectic A organisation is demonstrated in the lower temperature domain, which was hitherto understood as a cholesteric phase. A structural model for this phase is proposed on the basis of the analysis of the anisotropic scattering of stretched fibers. Our results also suggest that the observed glass transition is indeed a rather complex phenomenon, which seems to involve not only the freezing of the main chains, but also smectic correlations at the side-chain level. Moreover, the calorimetric study indicates that, notwithstanding the conservation of the processed film's optical properties, low kinetic reorganisations occur at room temperature. 相似文献
46.
Summary The synergids and other cells of mature, unpollinated pearl millet ovaries were investigated using: (1) freeze-substitution fixation in conjunction with scanning electron microscope observations and energy-dispersive X-ray microanalysis to localize total calcium (Ca) and other elements, and (2) antimonate precipitation to selectively localize loosely sequestered, exchangeable calcium (Ca++). In freeze-fixed ovaries, the synergid cells, ovary wall, nucellus, and other regions of the ovary displayed, respectively and relatively, extremely high, high, moderate, and low levels of Ca. In antimonate-fixed ovaries, Ca-containing antimonate precipitates exhibited similar distribution patterns. In ovaries fixed using the conventional 2% (w/v) antimonate in fixatives, the synergids were disrupted due to precipitate overload. In the ovary wall, precipitates were mainly located in the intercellular spaces. Some precipitates were observed at the micropyle and along the outer ovule integument, associated with diffuse extracellular material, and in the cell walls of nucellar cells proximal to the micropyle. Examination of precipitate distribution inside the synergids was possible in ovaries fixed using 0.5% (w/v) antimonate in the fixatives. Cytoplasmic organelles of all synergids examined exhibited variable states of disintegration. The amount of precipitates associated with the degenerated organelles appeared to be proportional to the degree of their degeneration. Distinct precipitates were localized in contiguous regions of the nucellar cells fused with the embryo sac, the micropylar half of the embryo sac wall, and the filiform apparatus. The results are discussed in relation to the involvement of Ca++ in mediating the functions of synergid cells during fertilization in angiosperms.On Specific Cooperative Agreement 58-43YK-8-0026 with the Department of Biochemistry, University of Georgia, Athens, GA 30602, USA 相似文献
47.
Disulfide bridges in tomato pectinesterase: variations from pectinesterases of other species; conservation of possible active site segments. 总被引:3,自引:0,他引:3
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Analysis of tomato pectinesterase by carboxymethylation, with and without reduction, shows that the enzyme has two intrachain disulfide bridges. Analysis of fragments obtained from the native enzyme after digestion with pepsin identified bridges connecting Cys-98 with Cys-125, and Cys-166 with Cys-200. The locations of disulfide bridges in tomato pectinesterase are not identical to those in three distantly related pectinesterases (18-33% residue identities) from microorganisms. However, one half-Cys (i.e., Cys-166) position is conserved in all four enzymes. Sequence comparisons of the overall structures suggest a special importance for three short segments of the entire protein. One segment is at the N-terminal part of the tomato pectinesterase, another in the C-terminal portion near the distal end of the second disulfide loop, and the third segment is located in the central part between the two disulfide bridges. The latter segment, encompassing only 40 residues of the entire protein, appears to high-light a functional site in a midchain segment. 相似文献
48.
Crystal structure of the Y52F/Y73F double mutant of phospholipase A2: increased hydrophobic interactions of the phenyl groups compensate for the disrupted hydrogen bonds of the tyrosines. 总被引:3,自引:1,他引:2
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C. Sekharudu B. Ramakrishnan B. Huang R. T. Jiang C. M. Dupureur M. D. Tsai M. Sundaralingam 《Protein science : a publication of the Protein Society》1992,1(12):1585-1594
The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 ester bond of membrane phospholipids. The highly conserved Tyr residues 52 and 73 in the enzyme form hydrogen bonds to the carboxylate group of the catalytic Asp-99. These hydrogen bonds were initially regarded as essential for the interfacial recognition and the stability of the overall catalytic network. The elimination of the hydrogen bonds involving the phenolic hydroxyl groups of the Tyr-52 and -73 by changing them to Phe lowered the stability but did not significantly affect the catalytic activity of the enzyme. The X-ray crystal structure of the double mutant Y52F/Y73F has been determined at 1.93 A resolution to study the effect of the mutation on the structure. The crystals are trigonal, space group P3(1)21, with cell parameters a = b = 46.3 A and c = 102.95 A. Intensity data were collected on a Siemens area detector, 8,024 reflections were unique with an R(sym) of 4.5% out of a total of 27,203. The structure was refined using all the unique reflections by XPLOR to a final R-factor of 18.6% for 955 protein atoms, 91 water molecules, and 1 calcium ion. The root mean square deviation for the alpha-carbon atoms between the double mutant and wild type was 0.56 A. The crystal structure revealed that four hydrogen bonds were lost in the catalytic network; three involving the tyrosines and one involving Pro-68. However, the hydrogen bonds of the catalytic triad, His-48, Asp-99, and the catalytic water, are retained. There is no additional solvent molecule at the active site to replace the missing hydroxyl groups; instead, the replacement of the phenolic OH groups by H atoms draws the Phe residues closer to the neighboring residues compared to wild type; Phe-52 moves toward His-48 and Asp-99 of the catalytic diad, and Phe-73 moves toward Met-8, both by about 0.5 A. The closing of the voids left by the OH groups increases the hydrophobic interactions compensating for the lost hydrogen bonds. The conservation of the triad hydrogen bonds and the stabilization of the active site by the increased hydrophobic interactions could explain why the double mutant has activity similar to wild type. The results indicate that the aspartyl carboxylate group of the catalytic triad can function alone without additional support from the hydrogen bonds of the two Tyr residues. 相似文献
49.
K. Harata M. Muraki Y. Hayashi Y. Jigami 《Protein science : a publication of the Protein Society》1992,1(11):1447-1453
The three-dimensional structure of a modified human lysozyme (HL), Glu 53 HL, in which Asp 53 was replaced by Glu, has been determined at 1.77 A resolution by X-ray analysis. The backbone structure of Glu 53 HL is essentially the same as the structure of wild-type HL. The root mean square difference for the superposition of equivalent C alpha atoms is 0.141 A. Except for the Glu 53 residue, the structure of the active site region is largely conserved between Glu 53 HL and wild-type HL. However, the hydrogen bond network differs because of the small shift or rotation of side chain groups. The carboxyl group of Glu 53 points to the carboxyl group of Glu 35 with a distance of 4.7 A between the nearest carboxyl oxygen atoms. A water molecule links these carboxyl groups by a hydrogen bond bridge. The active site structure explains well the fact that the binding ability for substrates does not significantly differ between Glu 53 HL and wild-type HL. On the other hand, the positional and orientational change of the carboxyl group of the residue 53 caused by the mutation is considered to be responsible for the low catalytic activity (ca. 1%) of Glu 53 HL. The requirement of precise positioning for the carboxyl group suggests the possibility that the Glu 53 residue contributes more than a simple electrostatic stabilization of the intermediate in the catalysis reaction. 相似文献
50.
Direct observation by X-ray analysis of the tetrahedral "intermediate" of aspartic proteinases. 总被引:1,自引:1,他引:0
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B. Veerapandian J. B. Cooper A. Sali T. L. Blundell R. L. Rosati B. W. Dominy D. B. Damon D. J. Hoover 《Protein science : a publication of the Protein Society》1992,1(3):322-328
We report the X-ray analysis at 2.0 A resolution for crystals of the aspartic proteinase endothiapepsin (EC 3.4.23.6) complexed with a potent difluorostatone-containing tripeptide renin inhibitor (CP-81,282). The scissile bond surrogate, an electrophilic ketone, is hydrated in the complex. The pro-(R) (statine-like) hydroxyl of the tetrahedral carbonyl hydrate is hydrogen-bonded to both active-site aspartates 32 and 215 in the position occupied by a water in the native enzyme. The second hydroxyl oxygen of the hydrate is hydrogen-bonded only to the outer oxygen of Asp 32. These experimental data provide a basis for a model of the tetrahedral intermediate in aspartic proteinase-mediated cleavage of the amide bond. This indicates a mechanism in which Asp 32 is the proton donor and Asp 215 carboxylate polarizes a bound water for nucleophilic attack. The mechanism involves a carboxylate (Asp 32) that is stabilized by extensive hydrogen bonding, rather than an oxyanion derivative of the peptide as in serine proteinase catalysis. 相似文献